To maintain genomic integrity, accurate DNA replication and chromosome segregation are under tight control during cell division. Recent studies implicate Cdh1 as a tumor suppressor essential in maintaining genomic integrity.15,16
There are several lines of evidence in support of this idea. First, Cdh1 is a target tightly regulated by genomic stress and prominently participates in the DNA damage response. Cellular exposure to UV radiation triggers proteolysis of Cdh1, whereas ionizing radiation causes its phosphorylation.40,41
Interestingly, both phosphorylation of Cdh1 and degradation of Cdh1, induced by different DNA-damaging agents, result in a similar effect: the abrogation of cyclin B1 degradation, which is believed to increase Cdk1 activity and enhance apoptosis upon genotoxic stress.41
In support of this, depletion of Cdh1 by gene knockout results in loss of checkpoint function in response to ionizing radiation and enhances the cellular susceptibility to apoptosis, whereas overexpression of non-degradable Cdh1 delays UV radiation-induced apoptosis.41
Another likely consequence of the inactivation of Cdh1, either by phosphorylation or by proteolysis, is to allow the onset of the DNA damage response. Our recent work identified Rad17 as a novel substrate of APC/Cdh1.7
Rad17 was initially identified in fission yeast as a DNA damage checkpoint protein and as an important component of the PCNA-like checkpoint-loading complex.42
The inactivation of Cdh1 after genotoxic stress leads to the accumulation of Rad17 protein. And accumulated Rad17 provides a platform for loading the 9-1-1 checkpointsliding clamp and Claspin after its phosphorylation by ATR.42,43
Recently, Claspin itself has been identified as a novel substrate of APC/Cdh1.8,11
Since the loss of Cdh1 leads to increased Claspin abundance, it is reasonable to assume that elevated Claspin levels will eventually activate the Chk1/p53 pathway to create a DNA damage-like response.8
Following the completion of cellular checkpoint activation after exposure to UV radiation, Rad17 undergoes proteolysis. The destruction of Rad17 results in loss of the platform bridging Claspin to other members of the checkpoint complex.7
The dissociation of Claspin from the checkpoint complexes due to Rad17 proteolysis as well as Claspin degradation disrupts the interaction between ATR and Chk1, a likely mechanism to terminate checkpoint signaling ().
Genotoxic stress leads to cell cycle arrest at G1 or G2 phase to provide sufficient time for cells to repair damaged DNA. Cdh1 plays a role in coordinating the DNA damage response in the cell cycle as its various substrates are active players in both DNA damage response and cell cycle progression ().
Depletion of Cdh1 stabilizes its substrates, such as Cdc6, Prc1 and Geminin, which may contribute to genetic instability by inducing re-replication.31,44
Unscheduled elevation of cyclin A and cyclin B, seen in Cdh1-depleted cells, interferes with loading of the pre-RC.15,16
For instance, Cdh1-depleted mice show reduced Mcm4 and Mcm5 levels on chromatin, suggesting a defect in pre-RC formation in these cells.15
Meanwhile, as complete inhibition of Cdh1 may not be necessary to bypass the G1
/S transition, Cdh1 could also participate in regulating origin firing in early S phase.30
As mentioned above, Cdh1 regulates Skp2-mediated p27 and p21 degradation, E2F-dependent cyclin E transcription and Ets2-induced cyclin D expression.25,28,29,45
Cdh1-null cells show a high rate of chromosomal translocations.15,16
This observation can be explained by the premature entry into S phase due to the failure to inactivate the cyclin-Cdk complex in G1
phase. Unscheduled origin firing leads to slow replication from fewer origins. This results in increased stalled replication forks and numerous replication errors, which in turn will activate the p53/p21-dependent replication checkpoint.44
Occasionally, cells will escape the checkpoint and enter mitosis, leading to chromosome breakage or translocation through breakage-fusion-breakage cycles.15,45
Cdh1 is also required for the DNA damage checkpoint in G2
Upon genotoxic stress in G2
, Cdh1 activation by Cdc14B-mediated dephosphorylation leads to degradation of substrates Plk1, Cyclin A and Cyclin B and prevents mitotic entry, opening a time window for DNA damage repair.11
Recent studies also revealed a Cdc14B-independent mechanism of Cdh1 activation: p53/p21-induced reduction of Cdh1 inhibitor Emi1.46
Given that depletion of Cdh1 can activate the p53/p21 pathway in G1
phase, the levels of p21 and Cdh1 seems to be regulated by a positive feedback loop. Previous gene-targeting experiments have shown that disruption of Cdh1 circumvents the G2
/M arrest normally induced upon DNA damage. However, mitotic entry is still delayed in Cdh1-null cells, indicating the existence of additional DNA damage checkpoints in G2
Genotoxic stress can activate Cdh1 in mitosis too. Under normal circumstances, Cdh1 is inhibited by phosphorylation by Cdk1 and polo kinase.20
DNA damage, however, leads to ATR-mediated inhibition of kinase activity of Cdk1 and polo kinase and thus the activation of Cdh1 in mitosis. Cdh1 targets Cin8/Kip1 and Ase1 to regulate spindle body elongation in early mitosis, while Cdh1-mediated Cyclin B and Cdc20 degradation in anaphase onset prevents the degradation of securin and thus blocks mitotic progression at the metaphase-anaphase transition.12,47
It is noteworthy that securin and Sgo1 are also substrates of Cdh1, suggesting that unscheduled or continuous Cdh1 activation may lead to premature anaphase onset.48,49
This also contributes to the aneuploidy observed in Cdh1-null cells.15
Downregulation of Cdh1, on the other hand, results in the accumulation of its substrates, including Aurora A, Plk1, cyclin A, cyclin B and Cdc20, which are part of the chromosomal instability signature in various murine and human tumors.15,16,50
Their accumulation in murine and human cells leads to genomic instability as evidenced by centrosome aberrations, multipolar mitosis, anaphase bridges and micronuclei.15,16
This is consistent with cultured Cdh1-null MEFs showing substantial numerical and structural chromosomal aberrations.15
Aurora kinases stabilized by Cdh1 inactivation could play a role in observed defects in anaphase spindle organization and premature cytokinesis, which can lead to polyploidy, supernumerary centrosomes and multipolar mitosis.13,15,16
This is consistent with the observed phenotype of Aurora A and Plk1 overexpression.51
Failed degradation of additional substrates, such as Cin8/Eg5 and Kip1 or Ase1/Prc1,12,13
may contribute to spindle and cytokinesis defects.
Besides its role in the regulation of the DNA damage checkpoint, APC/C has also been implicated in DNA repair, where APC/Cdh1 targets thymidine kinase 1 (TK1), a key cytosolic enzyme in the salvage pathway for dTTP synthesis, and ribonucleotide reductase R2, a critical enzyme that governs deoxynucleoside triphosphate biogenesis, for degradation. This suggests a key role of Cdh1 in regulating the dNTP pool, important in DNA replication as well as DNA repair.9,10
It is worth noting that an imbalanced dNTP pool may also dramatically increase mistakes in nucleotide incorporation and, consequently, the mutation rate.
APC/Cdh1 Modulates Signal Transduction and Regulates Cellular Differentiation
Considering the importance of Cdh1 in controlling the cell cycle, sensing growth signals and licensing DNA replication, it is not surprising that such a key component was also found to mediate receptor signaling and to regulate cellular differentiation. The following provide examples of such Cdh1-dependent signaling pathways ():
TGFβ plays both cytostatic and tumor enhancing roles based on the cellular context. In its cytostatic effect, upon stimulation with TGFβ, Smad3 and Smad2 translocate into the nucleus where they interact with both APC/C and SnoN, resulting in the APC/Cdh1-mediated degradation of SnoN that in turn leads to the activation of TGFβ downstream genes and growth inhibition of cultured cells.52,53
Furthermore, our group also demonstrated that TGFβ can trigger APC/Cdh1-dependent Skp2 degradation, which contributes to stabilization of p21 and p27 that is required for tumor suppression.54
NOTCH and JNK.
In Drosophila follicle cells, both Notch signaling and Cdh1 are required for the mitotic-to-endocycle transition.55
Cdh1 is expressed at the mitotic-to-endocycle transition in a Notch-dependent manner and mediates some effects of Notch signaling.55
A nuclear portion of the stress-activated kinase JNK is degraded by APC/Cdh1 during exit from mitosis and G1
phase, suggesting that JNK may be the target of Cdh1-mediated degradation induced by Notch signaling. Expression of a non-degradable JNK induces prometaphase-like arrest and aberrant mitotic spindle dynamics.56
Rb and E2F.
The retinoblastoma protein Rb interacts with APC/Cdh1 and controls the stability of p27 through targeting Skp2 for degradation. By specifically interacting with the hypophosphorylated (active) form of Rb, Cdh1 can regulate the activity of the E2F1 transcription factor.8,33
Meanwhile, E2F stimulates the transcription of early mitotic inhibitor-1 (Emi1)/Rca1, a pseudo-substrate inhibiting APC/Cdh1 activity.35
It has been well established that Ets2 is activated by the Ras-Raf-MAPK signaling pathway and stimulates cyclin D1 expression, which is the most prominent effect of this important pathway.29
Overactivation of Ras signaling causes oncogene-induced senescence in primary cells through Ets2-mediated increase of p16 expression.57
It is likely that Cdh1 can partially neutralize the overactivation of Ras signaling by degrading Ets2 and thus counteract its effects on p16. Cdh1 could also modulate Ras-induced phenomena, including de-differentiation, tumorigenesis and perhaps even metastatic diseases. Consequently, in normal human fibroblasts, depletion of Cdh1 results in premature senescence, whereas inactivation of both the p53 and Rb pathways by overexpressing SV40 LT-antigen partially reverses this phenotype.
The involvement of Cdh1 in apoptotic death was initially reported in B-lymphoma, where overexpression of Cdh1 dramatically increased cell susceptibility to natural killer cell (NK) cytotoxicity.58
A pro-apoptotic effect is also observed in cancer cells treated by UV radiation and in neurons in Alzheimer disease and stroke, sharing a common Cyclin B-Cdk2-triggered apoptotic program.41,59
This notion is also supported by the mitotic catastrophe of Cdh1-depleted tetraploid cells, which is mediated by the apoptotic pathway.51
It has recently been reported that Cdh1 targets Pfkfb3, a glycolysis-promoting enzyme, for degradation.60
Pfkfb3 was first identified to be responsible for the different capacities of neurons and astrocytes in tolerating oxidative stress. The accumulation of Pfkfb3 leads to the activation of the glycolysis pathway and promotes cell proliferation, a typical Warburg effect observed in cancer cells.60
Cdh1 directs cellular differentiation mainly at two steps. First, Cdh1 coordinates the balance between cell cycle exit and cell division. Second, Cdh1 promotes differentiation by activating differentiation-licensing factors through degradation of their transcriptional suppressors ().
Although Cdh1 has previously been suggested to be involved in mitotic exit, results from recent studies have emphasized its critical role in regulating the G1
phase and the quiescent G0
phase. Cdh1 is crucial to keep the mitotic cyclin activity low in early G1
and to regulate the length of G1
, leaving time for cell growth and differentiation.1
It has been shown that yCdh1 (FZR1
) mutant yeast fail to arrest under nutrient starvation, while dCdh1 (fzr) mutant flies undergo an extra embryonic epidermal cell division.6
Additionally, Cdh1 is not expressed during the early cell divisions of embryogenesis without distinctive G1
phase, whereas later expression in somatic cells correlates with a long G1
The most compelling evidence comes from the inducible deletion of cullin-like Apc2 in mouse liver cells causing quiescent G0
hepatocytes to re-enter the cell cycle without any additional proliferative stimuli.61
In particular, Cdh1 cooperates multiple pathways to maintain the G0/G1 state and to direct cell cycle exit: (1) TGFβ-induced SnoN degradation leads to de-repression of the p15 and p21 promoters; (2) Cdh1 controls Skp2 and Cks1 degradation and consequently the turnover of the CDK inhibitors p27 and p21 by SCFSkp2; (3) Cdh1 inhibits cyclin D transcription by degradation of Ets2; (4) Cdh1 completely eliminates mitotic cyclins, inducing degradation of the CDK1 activator Cdc25A and (5) APC/Cdh1 also directs the degradation of other positive regulators of cell proliferation (for example, Plk1 and Aurora A) and DNA replication (for example, Cdc6, Geminin, Tk1 and Tmpk).
The TGFβ-SnoN-p21/p15 axis is important for the TGFβ-induced neuron growth inhibition, axonal growth, morphogenesis and lens differentiation,62,63
while the Skp2-p27 and Ets2-Cyclin D1 pathways play a key role in myogenesis and differentiation of neuron and neuroblastoma SH-SY5Y.14,64
Moreover, it has been shown that Cdh1 can also regulate cellular senescence and the G1
balance via the APC/Cdh1-Ets2-p16 axis. Indeed, Cdh1-null MEFs proliferate poorly and enter senescence after only a few passages.26
Transcriptional activation of the differentiation licensing factors.
Studies based on cultured mouse myoblasts suggest that myogenesis could be regulated by APC/Cdh1 at different layers. Downregulation of Skp2 by APC/Cdh1 enhances withdrawal from cell cycle through stabilizing p21 and p27, which is necessary for cell differentiation. Moreover, time-dependent destruction of Myf5 was thought to be critical for facilitating myogenic fusion during the process of muscle differentiation.14
Inhibitor of differentiation/DNA binding (Id) proteins modulate cellular proliferation and differentiation in various cell types, such as neural or hematopoietic cells.65
While Id proteins are downregulated during cell cycle exit, overexpression of Id proteins in terminally differentiated cells will trigger cell cycle reentry. It has been reported that Id1, 2 and 4 might be targets of APC/Cdh1 in primary neurons and that Cdh1-dependent Id2 degradation inhibits axon growth and differentiation of neuronal stem cell into neurons.66
Id1 and Id2 also control the proliferation and differentiation in myeloid progenitors. Dysfunction of Id2 in mice or cultured cells caused by deletion or RNA interference induces lymphoid differentiation, whereas Id2 overexpression inhibits lymphoid and myeloid differentiation.65
These Id functions and their regulation by Cdh1 are consistent with the fact that Cdh1+/−
mice show plasmacytosis and myelodysplastic syndrome.15
Overall, APC/Cdh1 plays an important role in regulating cellular differentiation. However, present conclusions made based on cell culture systems need to be further validated in animal models.